Table I.
Steady State Kinetic Parameters for APH(2″)-IVa
| Substrate | kcat (s−1) | Km (μM) | kcat/Km (M−1 s−1) |
|---|---|---|---|
| 4,6-Disubstituted aminoglycosides | |||
| Kanamycin A | 0.92 ± 0.01 | 3.3 ± 0.3 | (2.8 ± 0.3) × 105 |
| Dibekacin | 1.23 ± 0.03 | 2.0 ± 0.3 | (6.2 ± 0.9) × 105 |
| Tobramycin | 1.80 ± 0.03 | 1.5 ± 0.1 | (1.2 ± 0.1) × 106 |
| Netilmicin | 0.64 ± 0.01 | <1 | >(6.4 ± 0.6) × 105 |
| Amikacin | 0.15 ± 0.01 | 98.0 ± 15 | (1.5 ± 0.3) × 103 |
| Isepamicin | 0.59 ± 0.02 | 17.0 ± 0.7 | (3.5 ± 0.2) × 104 |
| Arbekacin | 0.35 ± 0.01 | 18.0 ± 1.0 | (1.9 ± 0.1) × 104 |
| Nucleotide triphosphates | |||
| ATPa | 1.0 ± 0.1 | 310 ± 13 | (3.2 ± 0.3) × 103 |
| GTPa | 1.2 ± 0.1 | 330 ± 20 | (3.6 ± 0.4) × 103 |
All parameters were determined in 100 mM MES, pH 6.6, 80 mM NaCl, 10 mM MgCl2, and 20 mM KCl at 25°C. For the phosphorylation of aminoglycosides, ATP was used as the phosphate donor at a concentration of 2 mM.
a
Km and kcat of ATP and GTP were determined with a saturating concentration of kanamycin A (200 μM).